This Report involves work collected under protocols 01-M-0254 (NCT00024635 ); 08-M-0196 (NCT00759395); 08-M-0150 (NCT00697268); 14-M-0085 (NCT02122562); 07-M-0021 (NCT00397111); 14-M-0041 (NCT02049385); 07-M-0152 (NCT00472576), 09-M-N230; 15-M-0151 (NCT 02484456), 15-M-0188 (NCT02543983), and 19-M-0107 (NCT03973268). Results this past year: 1. Neurophysiological Changes Associated with Antidepressant Response to Ketamine Not Observed in a Negative Trial of Scopolamine in Major Depressive Disorder. This randomized, placebo-controlled, crossover trial examined the antidepressant efficacy of the muscarinic antagonist scopolamine in major depressive disorder subjects with more severe and refractory forms of major depressive disorder relative to previous reports. Participants included 23 medication-free major depressive disorder subjects currently experiencing a major depressive episode. Following a single-blind, placebo lead-in, participants were randomized to receive 2 counterbalanced blocks of 3 i.v. infusions of scopolamine (4 g/kg) and placebo in a double-blind manner. As assessed by both the Montgomery-Asberg Depression Rating Scale and Hamilton Anxiety Rating Scale, scopolamine had no significant antidepressant or anxiolytic effects relative to placebo. No significant drug vs placebo effects were seen in magnetoencephalography gamma power or brain-derived neurotrophic factor plasma concentrations, and brain-derived neurotrophic factor changes did not correlate with change in Montgomery-Asberg Depression Rating Scale score in response to scopolamine. These results do not support the efficacy of scopolamine for more severe or refractory forms of depression. 2. (2R,6R)-hydroxynorketamine exerts mGlu2 receptor-dependent antidepressant actions. Currently approved antidepressant drugs often take months to take full effect, and 30% of depressed patients remain treatment resistant. In contrast, ketamine, when administered as a single subanesthetic dose, exerts rapid and sustained antidepressant actions. Preclinical studies indicate that the ketamine metabolite (2R,6R)-hydroxynorketamine (2R,6R)-HNK is a rapid-acting antidepressant drug candidate with limited dissociation properties and abuse potential. We assessed the role of group II metabotropic glutamate receptor subtypes 2 (mGlu2) and 3 (mGlu3) in the antidepressant-relevant actions of (2R,6R)-HNK using behavioral, genetic, and pharmacological approaches as well as cortical quantitative EEG (qEEG) measurements in mice. Both ketamine and (2R,6R)-HNK prevented mGlu2/3 receptor agonist (LY379268)-induced body temperature increases in mice lacking the Grm3, but not Grm2, gene. This action was not replicated by NMDA receptor antagonists or a chemical variant of ketamine that limits metabolism to (2R,6R)-HNK. The antidepressant-relevant behavioral effects and 30- to 80-Hz qEEG oscillation (gamma-range) increases resultant from (2R,6R)-HNK administration were prevented by pretreatment with an mGlu2/3 receptor agonist and absent in mice lacking the Grm2, but not Grm3 -/-, gene. These findings highlight that (2R,6R)-HNK exerts antidepressant-relevant actions via a mechanism converging with mGlu2 receptor signaling and suggest enhanced cortical gamma oscillations as a marker of target engagement relevant to antidepressant efficacy. Moreover, these results support the use of (2R,6R)-HNK and inhibitors of mGlu2 receptor function in clinical trials for treatment-resistant depression either alone or in combination. 3. Antidepressant-relevant concentrations of the ketamine metabolite (2R,6R)-hydroxynorketamine do not block NMDA receptor function. Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine's antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK. 4. Functional Imaging of the Implicit Association of the Self With Life and Death. A critical need exists to identify objective markers of suicide ideation. One potential suicide risk marker is the Suicide Implicit Association Task (S-IAT), a behavioral task that uses differential reaction times to compare the implicit association between the self and death to the implicit association between the self and life. Individuals with a stronger association between the self and death on the S-IAT are more likely to attempt suicide in the future. To better understand the neural underpinnings of the implicit association between self and either life or death, a functional magnetic resonance imaging (fMRI) version of the S-IAT was adapted and piloted in healthy volunteers. An fMRI version of the S-IAT was administered to 28 healthy volunteers (ages 18-65, 14F/14M). Behavioral results were comparable to those seen in non-scanner versions of the task. The task was associated with patterns of neural activation in areas relevant to emotional processing, specifically the insula and right ventrolateral prefrontal cortex. Performance on the S-IAT fMRI task may reflect scores obtained outside of the scanner. In future evaluations, this task could help assess whether individuals at increased risk of suicide display a different pattern of neural activation in response to self/death and self/life stimuli. 5. Mouse, rat, and dog bioavailability and mouse oral antidepressant efficacy of (2R,6R)-hydroxynorketamine. (R,S)-ketamine has gained attention for its rapid-acting antidepressant actions in patients with treatment-resistant depression. However, widespread use of ketamine is limited by its side effects, abuse potential, and poor oral bioavailability. The ketamine metabolite, (2R,6R)-hydroxynorketamine, exerts rapid antidepressant effects, without ketamine's adverse effects and abuse potential, in rodents. We evaluated the oral bioavailability of (2R,6R)-hydroxynorketamine in three species (mice, rats, and dogs) and also evaluated five candidate prodrug modifications for their capacity to enhance the oral bioavailability of (2R,6R)-hydroxynorketamine in mice. Compared to intraperitoneal injection in mice, the relative oral bioavailability of (2R,6R)-hydroxynorketamine was 62%, which was not improved by any of the candidate prodrugs tested. Following oral administration, (2R,6R)-hydroxynorketamine readily penetrated the brain, with brain to plasma ratios between 0.67-1.2 in mice and rats. These results demonstrate that (2R,6R)-hydroxynorketamine has favorable oral bioavailability in three species and exhibits antidepressant efficacy following oral administration in mice.